Super-resolution is introduced in respect to conventional diffraction-limited resolution which is defined as a minimal distance (d) between two point-sources for which two images can be discerned as two separate irradiance maxima. There are several slightly different criteria of such discernibility. The classical diffraction-limited resolution can be represented as d=K×λ/(NA), where K=0.5, 0.61, 0.473, and 0.515. Here, the numerical aperture of the imaging system is represented by NA=no×sin θ, where no is the object-space refractive index and θ is the half of the objective's acceptance angle. Based on a solid-immersion concept, the top estimate for diffraction-limited resolution can be obtained by assuming that no is equal to the highest index in the system which is ns.
It is important to distinguish between the super-resolution in a fluorescent microscopy and in a label-free microscopy. The super-resolution in a fluorescent microscopy is very advanced area of studies. The recent 2014 Nobel prize in Chemistry was awarded “for the development of super-resolved fluorescent microscopy.” In the case of fluorescent microscopy, the biological or other objects are stained with fluorescent dye molecules or with the fluorophores. In some cases, brightly fluorescent quantum dots or other nanoscale objects can be used. In this type of microscopy, the actual “object” of imaging such as dye molecule, fluorophore or quantum dot is much smaller than the diffraction limit. This allows obtaining extraordinary detailed information about the object if additional techniques are used. There are many modifications of fluorescent microscopy. In all of these methods, the dye molecules, fluorophores or semiconductor nanocrystals are placed on the target structure by direct labeling techniques. The well-known example of such microscopies includes stimulate emission depletion (STED) method, but there are many other techniques such as photoactivated localization microscopy (PALM) microscopy, fluorescence photoactivation localization microscopy (FPALM), stochastic image reconstruction microscopy (STORM), and other methods using labeling or staining the tissue or samples. The resolution advantage of these methods is rooted in the fact that the actual “objects” are extremely small light sources, usually with dimensions on the scale of few nanometers or even smaller. Combining these small dimensions of the light sources with special types of nonlinear or bleaching effects allows obtaining better than diffraction-limited resolution in the fluorescent microscopy. It also simplifies measurement of the resolution of the system. The irradiance distribution of the image of such “point” objects represents so-called “point-spread function” (PSF). According to a Houston resolution criterion, the width of PSF is equal to the optical resolution of the system. So, measurement of the resolution is relatively simple in fluorescent microscopy because the point-objects are readily available. However, the fluorescence labeling has many disadvantages such as photobleaching and most importantly the need for labeling itself Methods and systems considered in this invention can be realized in combination with fluorescent microscopy, but mainly the proposed methods and systems belong to label-free microscopy.
For high-resolution applications, the label-free microscopy is more challenging and less developed compared to fluorescent microscopy. The ultimate freedom from fluorescent markers constitutes the goal of label-free microscopy. However, removing bright fluorescent objects immediately poses a main problem of the label-free microscopy—its low-contrast imaging mechanisms. Usually, the imaging is provided due to relatively weak light scattering mechanisms. Reduction of the size of the objects in the label-free microscopy makes them difficult to see. This means that it is challenging to discern small-scale objects on the background level of illumination inevitably presenting in microscopy. Another problem is that the measurement of the resolution becomes to be much harder problem in the label-free microscopy compared to the fluorescent microscopy. The textbook definitions of the optical resolution are made for idealized point sources. However, if the object is sufficiently small, it is not visible in the label-free microscopy. Generally, it means that more complicated methods of analysis of resolution based on images of finite-size objects are required in this area.
The approach to super-resolution imaging is based on using the optical near-fields since such fields can carry out very detailed information about the object. The problem is that the optical near-fields exponentially decay at very short distances (on the order of˜λ/2) from the object.
The near-field scanning optical microscopy (NSOM) gets around this problem in a complete and general way. The key point is that the photonic probe with a nanoscale hole in the metallic aperture or plasmonic probe with the nanoscale diameter is placed at very close distances from the object, where the non-propagating evanescent fields dominate. The resolution on the order of˜20 nm can be obtained by NSOM, however only by the expense of the transmitted intensity. The attenuation of the transmission to˜10−5 −10−6 is quite common for NSOM, especially for the smallest probe apertures. In addition, the probe can be easily damaged by the contact with the surface, and it needs sophisticated and precise setup for the sample surface scanning.
More recent and advanced approach to super-resolution imaging is based on using super-oscillatory lens. The ability to focus beyond the diffraction limit is related to the fact that band-limited functions are able locally to oscillate arbitrarily quickly, faster than their highest Fourier components, a phenomenon now known as super-oscillation. The resolution on the order o˜λ/6 has been claimed based on a discernibility of certain features in the optical images of these structures. It should be noted that more rigorous resolution treatment would require a convolution with the point-spread function. The limitations of this method are connected with a need in extremely precise fabrication and characterization equipment which is not likely to be available outside the leading research laboratories.
Another method of improving the resolution is based on illumination provided by the evanescent fields. However, for objects with complex shape, the use of this technique is not straightforward and it requires complicated image recovery algorithms which would make this method impractical in such cases.
One of the approaches to label-free super-resolution is based on using properties of plasmon-polaritons in metallo-dielectric structures, namely the property that they have much shorter wavelength compared to light waves with the same frequency propagating in air. This means that the plasmon-polaritons can carry out much more detailed spatial information about the object compared to conventional diffraction-limited optics. The idea of building super-resolution device is to provide a certain magnification of the image of the object using a plasmon-polariton medium (or a plasmon-polariton lens) where the detailed information about the object is preserved. The magnified image can be viewed by a conventional diffraction-limited microscope; however the information about the object details beyond the diffraction limit will be still observable due to the magnification provided by the plasmon-polariton medium. The limitations of this approach is that it works in a narrow range of frequencies, requires challenging fabrication, and it is usually applicable only for in-plane propagation of light.
Another approach is based on using far-field hyperlens. The hyperlens utilizes cylindrical or spherical geometry to magnify the subwavelength features of imaged objects so that these features are above the diffraction limit at the hyperlens output. Thus, the output of the hyperlens consists entirely of propagating waves, which can be processed by conventional optics. Near-field plasmonic super-lenses and 3D hyperlenses have been demonstrated with resolutions λ/3.6 and λ/2.6, respectively. These systems operate in narrow spectral ranges and are very difficult to fabricate. More recently, a non-resonant hyperlens was demonstrated in visible. It should be noted, however, that hyperlenses developed in all these works require a challenging fabrication. Most importantly, they are difficult to use with the objects which are not fabricated inside the imaging device. This means that the practical use of these structures for imaging biological and other similar samples is very limited at present time.
There arc other methods of creating label-free contrasts in microscopy based on the harmonic generation of the illumination or four-wave mixing processes through the nonlinear response of the sample. For instance, it has been shown that collagen can be nicely imaged through second harmonic generation (SHG).
In this regard, imaging by dielectric microspheres or microcylinders emerged as a surprisingly simple way of obtaining super-resolved images of nanoscale structures. The method implies bringing a dielectric microsphere in a contact position with investigated structure, so that the microsphere experiences the object's optical near-field and creates a magnified virtual image that can be viewed by a standard microscope at a certain depth inside the structure. Initially, the method has been demonstrated for micron-scale low-index (ns=1.46) silica spheres in air. At that time, it has been proposed that this technique would not work for high-index (ns>1.8) spheres as well as for spheres totally immersed in a liquid. So, the proposal to use high-index spheres totally submersed in a liquid or embedded inside elastomeric slabs was counterintuitive and inventive. Later, it was demonstrated that it was this proposal that turned out to be the most useful method for imaging biomedical samples. The label-free super-resolution microscopy has been realized for imaging of adenoviruses and mitochondria using liquid-immersed high-index microspheres. The fluorescent imaging through microspheres has been realized for stained biological structures.